Uncoveringthe hidden signsof organ transplantrejection

Researchers are discovering and testing biomarkers to help guide treatment and improve long-term outcomes for transplant patients

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On TV medical dramas, a common suspenseful story line follows a person in need of an organ transplant waiting to find a donor. A happy ending ensues when the organ arrives, packed in ice and ready for surgical insertion.

In brief

Thousands of organs are transplanted every year. Those organs are constantly under attack from the body’s immune system. Although short-term outcomes are good, long-term survival of transplanted organs remains below 60%. Molecular biomarkers in blood and urine can provide physicians with information about immune status and organ function. But physicians don’t yet know whether making treatment decisions on the basis of those biomarkers will improve outcomes.

To be sure, organ transplants save lives. More than 33,000 organs were transplanted in the U.S. in 2016, the most recent year for which data are available, according to the United Network for Organ Sharing. In the real world, though, after a transplant, a recipient’s immune system fights against the organ, recognizing it as foreign. As many as 15% of people receiving kidney transplants, for instance, experience acute rejection, meaning that the immune system sends in its troops and causes inflammation within the first year. For those patients, the story line isn’t tied up neatly.

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Rejection episodes like these are usually treatable and reversible. So “rejection” doesn’t necessarily mean the transplanted organ—or graft, as doctors like to call it—is lost. Tissue damage can occur during these episodes, however, and if it happens enough times or is not treated quickly enough, the organ fails.

To ensure prompt treatment, physicians need a way to monitor organ function and diagnose rejection. The only method available to them now is direct biopsy of the transplant. In the case of a kidney transplant, the typical schedule for routine monitoring is to take biopsies of the organ at one week, one month, three months, six months, one year, and yearly after that.

Rather than poke a needle into an organ to sample cells and tissues—a risky venture—doctors would prefer to noninvasively measure molecular markers in a person’s blood and urine to monitor recovery. These markers, typically nucleic acids and proteins, could help indicate when a transplanted organ has started to fail, help guide treatments, minimize the number of biopsies needed, and ultimately improve transplant success rates.

The anatomy of rejection

Not all transplant rejection is the same. It can be divided into two major categories, distinguished by the mechanism through which it occurs and to a lesser extent by the length of time since transplant.

Acute rejection typically results from immune cells called T cells infiltrating the organ and causing local inflammation that damages the organ. To stave off acute rejection, patients take drugs that suppress immune function. If an episode happens anyway, it can be treated with high doses of steroids. Acute rejection episodes typically occur in the first year after transplant, but if a recipient stops taking the immunosuppressants, they can occur months or even years after the transplant.

Chronic rejection, in contrast, is primarily an antibody-mediated process. During chronic rejection, the person’s immune system continually attacks the transplanted organ. Eventually the damage builds up enough that the organ stops working. A patient experiencing chronic rejection doesn’t display symptoms until the situation is too advanced to do anything about it. No treatment for the condition yet exists.

Biomarkers that indicate how a person’s immune system is functioning could help guide doctors in prescribing the correct dose of immunosuppressants for individual transplant patients to help ward off acute rejection. And they might someday help develop a treatment for chronic rejection.

“Most transplants are treated the same,” says Peter S. Heeger, a transplant immunologist at Icahn School of Medicine at Mount Sinai. At most centers, he says, patients receive therapy to dial down the immune system at the time of the transplant. After the transplant, they receive multiple immunosuppressant drugs.

“Because we don’t have a tool to measure immune function, we usually give all patients the same amount of medication,” says Jamil R. Azzi, an expert in kidney transplants at Brigham & Women’s Hospital and Harvard Medical School.

Physicians typically “wait six months and slowly lower the dose of one of the drugs. They don’t really measure anything. They just wait and see what happens,” Heeger says.

Such an approach means that some people receive more drug than they need, whereas others receive less. Both of these options can have negative consequences. Too much drug suppresses the immune system too much, leaving a patient susceptible to infections. Side effects of overprescribing immunosuppressants are a major factor in the premature death of transplant recipients. At the other extreme, too little drug can pave the way for rejection and organ loss. Physicians want to avoid both of these situations and tailor the amount of immunosuppressants that patients receive.

But the only way that physicians currently have for determining whether they should adjust a patient’s drug dose is to take a biopsy. “There are complications that come from putting a needle into an organ with lots of vessels,” Azzi says. Beyond the risk of bleeding, there’s the time, expense, and patient anxiety to contend with, he says.

“We’re hoping for a noninvasive test that we can do frequently,” Azzi says. “If there’s any signal with this test showing higher probability for ongoing rejection—even mild—then we biopsy.” Biomarkers that signal rejection could offer such a diagnostic.

“With the advancement of technology, we envision that we’ll be able to develop biomarkers that not only tell us the diagnosis but also the severity of the disease,” Azzi says.

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Today's biomarkers

The long road to transplant biomarkers

Biomarkers need to go through many stages before they can be used in clinical practice. In the case of transplant rejection, candidate biomarkers are still waiting for prospective clinical trials. After those trials, the biomarkers will still need to go through commercialization and final implementation. None of the transplant biomarkers have yet reached that stage.

In the past, discovery took five to 10 years. Newer technologies allow it to happen in months to several years.

Validation(Test to ensure biomarkers agree across platforms and among research groups at multiple centers)

Existing samples from patient cohorts with known end points allow validation to happen quickly. In the absence of such samples, multicenter trials need to be performed, which can take several years.

Clinical trial(Conduct prospective, multicenter trials in which biomarkers are used to make treatment decisions)

Can take several years.

Commercialization and implementation

Unknown

Current biomarkers fall short of that goal. For example, in the case of kidney function, physicians for decades have been limited to creatinine as a biomarker. Inside the body, the level of creatinine—a breakdown product of muscle—remains fairly constant. It’s the kidneys’ job to filter the chemical from the body, so changes in the amount of creatinine in urine can signal problems with the organs’ function. Valeria Mas, director of the Translational Genomics Transplant laboratory at the University of Virginia, says creatinine is an inaccurate biomarker. Yes, it can be detected noninvasively in a patient’s urine without doing a biopsy. But its levels increase only after major damage to the kidneys has occurred.

“By the time the creatinine is up, a few weeks, months, or years have passed, and now you have a severely scarred kidney,” Azzi says. Just as a fire can smolder and burn without producing a flame, there can be “smoldering rejection that we’re not detecting with current biomarkers, leading to chronic damage,” he says.

Because creatinine has been the best biomarker available for kidney dysfunction for the past few decades, “we’re not making the progress that other fields like cancer are making,” Mas says. That field has been more successful in identifying appropriate biomarkers.“But now I’m optimistic that we’re on the right path,” she adds.

Mas’s team is hunting for biomarkers that signal immune status and graft response to injury. The researchers are establishing a composite score calculation that includes clinical characteristics such as donor age and recipient sex along with messenger RNA and microRNA molecular markers that can be used to determine whether the organ is functioning properly. “The composite score represents a holistic approach rather than a single marker,” Mas says. “The human transplant model is too complex to assume that a single biomarker will provide accurate information about the graft status.”

One of the furthest advanced panels of RNA biomarkers comes from the lab of Manikkam Suthanthiran at Weill Cornell Medical College. The scientists there test for acute rejection in kidney transplant patients by quantitatively measuring the expression of genes in cells isolated from the recipients’ urine.

The team carried out a multicenter trial with a set of eight genes (N. Engl. J. Med. 2013, DOI: 10.1056/NEJMoa1215555). A statistical analysis of the data revealed that the scientists could use the expression levels of as few as three of the genes to calculate an acute rejection score.

They used the score to correctly identify patients who went on to have an acute rejection episode. “About 20 to 40 days before rejection, you can see that the score starts to go up,” Suthanthiran says. “The molecular rejection seems to precede the clinical rejection.”

The biomarker panel could save patients from having unnecessary biopsies. In the future, Suthanthiran wants to conduct trials to see whether the information from the biomarkers can be used to make decisions about the amount of immunosuppressants a patient receives.

Azzi’s team is assessing whether extracellular vesicles in urine called exosomes can be used to detect kidney transplant rejection (ACS Nano 2017, DOI: 10.1021/acsnano.7b05083). These exosomes are shed by cells and therefore reflect the content and constituents of those cells. For example, the protein CD3, a cell surface receptor, is unique to T cells. If CD3 appears in exosomes in urine, T cells have infiltrated the transplanted kidneys, Azzi says. “This means you have T cells attacking the kidney” and acute rejection has begun.

Azzi plans to use exosomes to identify other proteins or genes that can be combined in a biomarker panel to monitor the organ. “We envision not only diagnosing rejection. We want to diagnose different kinds of rejection. We want to diagnose the severity of rejection. We want to diagnose the state of the transplanted kidney itself.”

Transplant stats

The odds for long-term transplant success remain stubbornly low, as shown for some of the most-transplanted organs.

33,610

Number of all organ transplants in the U.S. in 2016

19,060

Number of kidney transplants in the U.S. in 2016

55%

Proportion of kidneys transplanted in the U.S. in 2006 that survived at least 10 years

7,841

Number of liver transplants in the U.S. in 2016

56%

Proportion of livers transplanted in the U.S. in 2006 that survived at least 10 years

3,191

Number of heart transplants in the U.S. in 2016

58%

Proportion of hearts transplanted in the U.S. in 2006 that survived at least 10 years

2,327

Number of lung transplants in the U.S. in 2016

30%

Proportion of lungs transplanted in the U.S. in 2006 that survived at least 10 years

In search of validation

One hurdle to identifying protein-based biomarkers in tissue samples is that the groups working in this area use different proteomics methods to carry out the screening, says Katrin Kienzl-Wagner, a surgeon at Medical University of Innsbruck who also conducts research to identify biomarkers of transplant rejection.

“Everybody is applying a slightly different experimental setup, so it makes it difficult to compare data from different groups,” Kienzl-Wagner says. “Maybe the biggest hurdle is that the patient populations in the proteomics studies that have been done so far are rather small. If you really want to validate a marker, you need large study cohorts, which would mean that all transplant centers from Europe or the U.S. or even worldwide would have to do the same experiment.”

Transplant stats

The odds for long-term transplant success remain stubbornly low, as shown for some of the most-transplanted organs.

33,610

Number of all organ transplants in the U.S. in 2016

19,060

Number of kidney transplants in the U.S. in 2016

55%

Proportion of kidneys transplanted in the U.S. in 2006 that survived at least 10 years

7,841

Number of liver transplants in the U.S. in 2016

56%

Proportion of livers transplanted in the U.S. in 2006 that survived at least 10 years

3,191

Number of heart transplants in the U.S. in 2016

58%

Proportion of hearts transplanted in the U.S. in 2006 that survived at least 10 years

2,327

Number of lung transplants in the U.S. in 2016

30%

Proportion of lungs transplanted in the U.S. in 2006 that survived at least 10 years

Most biomarkers are being identified from gene expression profile or peptide-based proteomics studies. Neil L. Kelleher, a mass spectrometrist at Northwestern University, is instead working with doctors at Northwestern’s Comprehensive Transplant Center to identify proteoforms in blood that can serve as biomarkers of acute rejection.

A proteoform is a specific molecular form of a protein produced from a human gene. For example, a proteoform could be a protein that’s had a particular functional group, added to it—a posttranslational modification. Or it could be a protein with a slight sequence variation compared with the gene from which it was produced. These seemingly small differences can dictate significant changes in protein function and correlate strongly with organ rejection. But they can be lost during the processing steps of conventional proteomics methods that involve digesting whole proteins into peptides. So Kelleher’s team searches for proteoform biomarkers with so-called top-down proteomics methods, which allow the researchers to work with intact proteins.

Josh Levitsky, a liver transplant physician at Northwestern’s Comprehensive Transplant Center who collaborates with Kelleher, emphasizes that they’ve been using frozen but still viable immune cells. “We’re seeing what proteins and proteoforms are being produced at the time of rejection,” he says. “It feels most representative of what’s going on physiologically.”

They found that most of the proteoforms expressed at different levels in healthy transplant patients and in patients who had experienced acute rejection were variants that differed from their expected sequence. The most significant of the proteoforms were variants of a protein known as CXCL4, which is thought to play roles in inflammation and wound repair. The variants were more abundant in healthy recipients, suggesting they might be protective (Am. J. Transplant. 2017, DOI: 10.1111/ajt.14359).

Rejection biomarkers are typically identified for a specific organ type. But by doing a meta-analysis of eight independent data sets collected from transplant patients, Minnie Sarwal, now at the University of California, San Francisco, and coworkers identified a common rejection module, or CRM: a set of 11 genes whose expression is elevated during acute rejection in four organ types—kidney, lung, heart, and liver (J. Exp. Med.2013, DOI: 10.1084/jem.20122709).

One drawback is that the data sets came from tissue biopsy analysis rather than blood or urine analysis, so the CRM might not ultimately apply to the type of diagnostics doctors seek. Still, when the researchers tested their CRM against a set of kidney tissue samples, they found that the samples from organs that had been acutely rejected correlated with a higher CRM score (PLOS One 2015, DOI: 10.1371/journal.pone.0138133).

Driving toward the clinic

Researchers agree that a major hurdle to getting transplant biomarkers to the clinic is a lack of funding. A perceived lack of funding notwithstanding, the U.S. National Institutes of Health is actually supporting clinical trials through the Clinical Trials in Organ Transplantation, or CTOT, consortium. Sponsored by the National Institute of Allergy & Infectious Diseases, CTOT has a mission to run studies that improve short-term and long-term outcomes for transplant recipients. CTOT, established in 2004, is now in its third funding cycle. So far, CTOT has doled out more than $135 million. Since its inception, the consortium has undertaken 21 clinical trials, 12 of which are now closed, eight of which are currently active, and one of which is in development.

Part of the goal is to identify reliable biomarkers and then, once you have them, to use them to treat people on the basis of the presence or absence of the biomarker,” says Mount Sinai’s Heeger, who is a member of CTOT’s steering committee.

For example, Suthanthiran’s gene panel was tested in a CTOT trial. And the CRM developed by Sarwal and colleagues will be tested as a biomarker panel in future NIH-funded clinical trials that are currently in the planning stages, says Tara K. Sigdel, a member of Sarwal’s group.

The aim of CTOT’s studies varies depending on the transplanted organ being considered. For instance, the outcomes for lung transplants are significantly worse than for other transplants, Heeger says. “We have to understand and fix that,” he says.

With that goal in mind, one CTOT trial is an observational study of about 800 lung transplant patients. The study is collecting blood and biopsy samples, as well as fluid obtained from rinsing lung tissue. “They’re looking for what are the best markers and trying to understand why the organs are rejected,” Heeger says.

But ultimately, physicians need biomarkers that can improve outcomes. For that to happen, trials need to be done in which some patients are treated on the basis of the results of biomarker tests and other patients receive the current standard of care.

“We now have a series of biomarkers that seem to be reproducibly able to detect acute injury to the transplant,” especially for the kidneys, Heeger says. “What we need to do now is design studies to test whether treating a patient based on those biomarkers is going to change the outcomes.”

And one of the biggest needs is improving the long-term outcomes. Short-term organ-survival rates for all organs have improved, but 10-year survival rates have remained stubbornly stuck below 60%. For lung transplants, that number is lower—closer to 30%.

To try to bring those survival rates up, the University of Virginia’s Mas wants researchers to focus less on acute rejection and more on the process that leads to chronic rejection. Even though doctors prescribe strong immunosuppressants, “the immune system always finds a way to respond to the graft, even when we cannot see any clinical manifestation.” The focus needs to be on avoiding that immune response that doctors aren’t seeing clinically but is leading to chronic rejection, she adds. “Monitoring the graft is going to be the solution.”

Heeger agrees that more attention needs to be paid to long-term outcomes, but “it’s hard to do studies that are 10 years long,” he says. Instead researchers need a surrogate end point.

“Let’s say I want to know who’s going to lose their graft in 10 years and then identify a biomarker that’s going to tell me that. I can’t wait 10 years to find out.” But if researchers can find a marker at the two-year point that accurately predicts what will happen after 10 years, they may be able to adjust treatment to ward off graft loss.

But the hurdle—and perhaps a reason that there have been so few studies—is that even with a biomarker for chronic rejection, which is usually antibody based, no good treatments exist, Heeger says.

“We don’t have good drugs to ward off antibody responses,” he adds. “And people who develop antibodies often end up doing worse.”

As eager as he is for biomarkers to succeed, Azzi says that as a physician he won’t use any biomarker unless he believes that it has predictive value and will improve outcomes. “I need more data to do that,” he says.

“We have our work cut out for us,” Heeger says. “It’s always nice to identify new things, but we’ve already identified and validated a number of biomarkers. It’s time to determine if acting on them improves outcomes.”